T.70. Different Mechanisms of Immunosuppression Caused by Cyclosporine A (CsA) and FK506

S70 syngeneic splenocytes were administered as immune cell therapy (CT) prior to vascular endothelial growth factor receptor blockade (SU5416). Methods and Results: AT rats were given a single dose of SU5416 (SU) at a dose of 20 mg/kg s.c (AT + SU) group. Control AT was treated with vehicle only (DMSO). AT was given 20 × 106 spleen cells i.p. 7d prior to SU (CT). 2D-Echo, pulmonary hemodynamics and RV/LV + S ratio were assessed at 10d and 21d. Echo revealed decreased RV fractional shortening (p b 0.05) and PA acceleration time (pb 0.005) in AT + SU compared with CT and controls. CT prevented increase of RV systolic pressure compared to AT + SU (24.3 ± 0.35 vs. 43 ± 3.87 mmHg (P b 0.01) and of RV/LV + S (0.24 ± 0.03 vs. 0.38 ± 0.11 (Pb 0.001). RVH fetal gene expression (a-MHC, b-MHC, SERCA, BNP, SkACT, ANP) was consistent with pathologic stress pattern and was significantly attenuated by CT. Furthermore, CT reduced expression of TNFalpha, INFgamma, RANTES, IL-4 and increased expression of IL-10, and BMPR2. CT also reduced pulmonary infiltration of macrophages, mast cells, and B cells while increasing lung and blood expression of HOX11+, C-kit+, FOXP3 + cells. Conclusions: This is the first study to show that splenocytes administered as syngeneic immune CT prevents PH associated with RVH, RVF and inflammation. Given recent evidence that HOX11+ stem cells may be therapeutic for autoimmune conditions, it is possible that PH prevention is because of accumulating of HOX11+, C-kit+and FOXP3+ cells. doi:10.1016/j.clim.2009.03.203

T.69. T Cell Activation and Th2-related Gene Expression Correlate with Disease Activity in Glatiramer Acetate-treated Multiple Sclerosis Finn Sellebjerg1, Martin Krakauer1, Dan Hesse1, Henrik Lund2, Signe Limborg1, Helle Søndergaard1, Per Sørensen1. 1 Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark; 2Copenhagen University Hospital Hvidovre, Hvidovre, Denmark Glatiramer acetate (GA), a synthetic immunomodulatory substance generated by random polymerization of glutamate, lycine, alanine and tyrosine, is used for the treatment of multiple sclerosis (MS). GA activates specific T cells, induces regulatory T cells, a T helper type 1 (Th1) to Th2 cytokine secretion shift, and changes the activation and cytokine secretion of antigen presenting cells (APCs). The extent to which these effects correlate with disease activity is uncertain. We studied gene expression in whole blood mononuclear cells (Affymetrix Human Genome Focus GeneChip), cytokine and transcription factor gene expression in whole blood (real-time PCR analysis) and CD4 + T cells, monocytes and dendritic cells (flow cytometry) in untreated MS patients and patients treated with GA for 3 months, 6 months or more than 6 months (long-term treated). Disease activity was assessed by magnetic resonance imaging. GA treatment reduced the expression of some markers of T cell activation after 6 months. Long-term treatment reduced the expression of many Th1-and Th17-associated molecules, increased regulatory T cell marker expression on CD25hi CD4 + T cells, and decreased costimulatory molecule expression on APCs. Markers of CD4 + T cell activation correlated

Abstracts positively and markers of Th2 activation correlated negatively with disease activity during treatment. APC activation markers, regulatory T cell markers and the expression of genes identified as GA-regulated by DNA array analysis did not correlate with disease activity. We conclude that decreased expression of T cell activation markers is the earliest observable effect in GA-treated MS patients. Markers reflecting the T cell activation and Th1/Th2 balance correlate with disease activity, suggesting that these are the most therapeutically relevant effects of treatment with GA. doi:10.1016/j.clim.2009.03.204

T.70. Different Mechanisms of Immunosuppression Caused by Cyclosporine A (CsA) and FK506 Thomas Giese, Claudia Sommerer, Martin Zeier, Stefan Meuer. University Hospital Heidelberg, Heidelberg, Germany Calcineurin inhibitors (CNI) are the cornerstones of most current immunosuppressive protocols used for transplanted patients. The incidence of acute rejection episodes dropped remarkably after the introduction of CNI into clinical practice. The key mechanism of both CsA and FK506 is the inhibition of calcineurin, leading to the prevention of NFAT translocation into the nucleus of T-cells with a subsequent block of transcription of crucial cytokine genes. However, the two drugs exert different clinical activities as exemplified by the ability of FK506 not only to prevent but also to treat acute rejections. Both drugs exert a different safety profile. Inhibition of calcineurin by FK506 occurs in vitro at the same or even higher dose as for CsA, however, the magnitude of clinical and experimental (cell culture) immunosuppression is higher, indicating that FK506 may act in a calcineurin-independent way. Recently we introduced an assay for monitoring the immunosuppressive effects of CsA. Based on the quantitative analysis of NFAT-dependent gene expression in whole blood before and after drug intake, this test was used to perform the pharmacodynamic analysis of NFAT-inhibition of patients treated with FK506. Surprisingly, in the majority of patients with FK506 peak levels below 20 mg/l expression of NFAT-regulated genes was only marginally inhibited, suggesting that an alternative mechanism is responsible for the FK506-mediated immunosuppression. This effect was also observed on the level of cytokine release, indicating that FK506 does not inhibit protein translation. Therefore, we suggest that transplant rejection can be prevented by FK506 independent of NFAT-dependent cytokine inhibition. doi:10.1016/j.clim.2009.03.205

T.71. Effects of Interferon-beta Treatment on T Cell Signature Cytokines and Transcription Factors in Multiple Sclerosis Lars Börnsen, Dan Hesse, Martin Krakauer, Per Sørensen, Finn Sellebjerg. Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark